Group A Streptococcus (GAS) is a human pathogen of global importance that causes self-limiting infections at the throat (pharyngitis) or skin (impetigo), leading to ~750 million infections per year. GAS is associated with high rates of morbidity and mortality due to autoimmune and invasive disease. Acute rheumatic fever (ARF) follows an inadequately treated GAS throat infection by a so-called rheumatogenic strain, and can lead to rheumatic heart disease via autoimmune attack of heart valves. The existence of distinct rheumatogenic and non-rheumatogenic strains of GAS has been long recognized, yet their unique properties remain elusive. Our preliminary data shows significant differences in the activity of a secreted cysteine protease (SpeB) for GAS recovered from patients with different diseases (impetigo > pharyngitis > ARF). These findings provide support for a novel hypothesis for a longstanding puzzle: That rheumatogenicity is, at least in part, a function of a GAS phenotype that is related (directly or indirectly) to SpeB activity. Importantly, SpeB is a key biomarker for the transcriptional program of the bacterial cell, as well as a virulence factor that acts by degrading human host proteins involved in defense. The overall goal is to delineate the molecular basis for differential SpeB activity among ARF- versus pharyngitis-associated GAS due to differences in transcriptional networks. The hypothesis to be tested is that GAS recovered from different diseases - ARF and pharyngitis - differ in their transcriptional programs. This is to be achieved by defining the transcriptomes of a select set of biologically diverse isolates of GAS, via RNA-Seq (Aim 1), and screening a large population of GAS strains in order to identify transcriptional signatures that distinguish clinically important sub-populations, via quantitative RT-PCR and cluster analysis (Aim 2). The proposal provides a broad approach for comparative transcriptomics on a bacterial population level. A long-term goal is to understand the molecular basis for distinct GAS diseases. Data supporting the hypothesis may uncover alterations in transcriptional pathways that affect not only speB expression, but also transcription of other genes which may have a direct role in triggering ARF. SpeB activity (or its absence) may directly contribute to ARF pathogenesis through (lack of) modulation of host and/or GAS extracellular proteins. Overall, this exploratory study may lead to more comprehensive future studies that advance our understanding of the molecular processes triggering ARF, improve experimental models for ARF, and better inform vaccine design and diagnostics.